Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 418302, 9 pages http://dx.doi.org/10.1155/2014/418302
Research Article Application of Ultrasound on Monitoring the Evolution of the Collagen Fiber Reinforced nHAC/CS Composites In Vivo Yan Chen,1 Yuting Yan,2 Xiaoming Li,3 He Li,2 Huiting Tan,2 Huajun Li,2 Yanwen Zhu,2 Philipp Niemeyer,4 Matin Yaega,4 and Bo Yu5 1
Department of Ultrasonic Diagnosis, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China The Second Clinical Medical College of Southern Medical University, Guangzhou 510282, China 3 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, China 4 Department of Orthopaedic surgery and Traumatology, Freiburg University Hospital, Freiburg, Germany 5 Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China 2
Correspondence should be addressed to Xiaoming Li; [email protected] and Bo Yu; [email protected] Received 31 October 2013; Accepted 4 March 2014; Published 14 April 2014 Academic Editor: Xiaowei Li Copyright © 2014 Yan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To date, fiber reinforce scaffolds have been largely applied to repair hard and soft tissues. Meanwhile, monitoring the scaffolds for long periods in vivo is recognized as a crucial issue before its wide use. As a consequence, there is a growing need for noninvasive and convenient methods to analyze the implantation remolding process in situ and in real time. In this paper, diagnostic medical ultrasound was used to monitor the in vivo bone formation and degradation process of the novel mineralized collagen fiber reinforced composite which is synthesized by chitosan (CS), nanohydroxyapatite (nHA), and collagen fiber (Col). To observe the impact of cells on bone remodeling process, the scaffolds were planted into the back of the SD rats with and without rat bone mesenchymal stem cells (rBMSCs). Systematic data of scaffolds in vivo was extracted from ultrasound images. Significant consistency between the data from the ultrasound and DXA could be observed (𝑃 < 0.05). This indicated that ultrasound may serve as a feasible alternative for noninvasive monitoring the evolution of scaffolds in situ during cell growth.
1. Introduction Cell-based bone tissue engineering has emerged as a promising alternative to traditional bone graft treatment [1]. Due to mineralized collagen fibers making up the microstructure of natural bone tissue [2], a biomimetic nanohydroxyapatite/collagen (nHA/Col) scaffold reinforced by mineralizing type I collagen fiber seems to be a very promising system for bone tissue engineering [3–5]. Hence the development of mineralized collagen fiber composites is in urgent need of a noninvasive, quantifiable, and systematic method to monitor the complex regenerate function and degradation process in vivo and in real time. Accurate in vivo data is needed for a complete understanding of the mineralized collagen fiber and guiding the scaffold design. Developing a simple and easy-to-use method to monitor regeneration process of the scaffolds is critical for bone tissue
engineering research. Current approaches for acquiring precise bone mineral density (BMD) value are mostly by dualenergy X-ray absorptiometry (DXA) or computed tomography (CT) [6]. However, for one thing, the scanning and image reconstruction procedures are of complex operations, in high consumption and with strong radiation. Thus it is difficult to meet the need of a long-term evaluation of dynamic tracking. For another, DXA and CT could only provide morphological information and are unable to achieve the exploration of bone microstructure, which is the determining factor of bone function independently of BMD [7, 8]. To date, measurements on bone constructs and some desired mechanical parameters mainly rely on destructive and timeconsuming histological and biochemical assay [9]. The substantial animal use, low repeatability, and difficulty of in vivo examination limit its application. There are also some budding nondestructive technologies. MR elastography (MRE)
2 could make an assessment of mechanical properties, but it is limited by a poor spatial resolution at 5 mm [10]. Optical coherence tomography (OCT), with high spatial resolution but low penetration capacity, was mainly used in evaluation of vascular scaffold currently [11]. Microcomputed tomography (𝜇CT) could evaluate the scaffolds systematically, but its use was limited by its high expense and equipment requirement [12]. Hence, there remains a significant need for noninvasive techniques to sequentially monitor the progress of tissue construct evolution in vivo without periodic animal sacrifice. Since the report about ultrasonic speed and attenuation in bone in 1975 [13], ultrasound was gradually developed to be a noninvasive, nonradiative diagnostic tool of bone. Physical parameters tested by ultrasound were capable of reflecting bone density, quality, and some other mechanical factors of cancellous bone [14, 15]. Consequently, ultrasonic technology has been widely used in analyzing children’s bone condition and osteoporosis in recent years and reveals a promising application [16]. However, ultrasound is rarely reported to evaluate scaffolds in bone tissue engineering. Recently, ultrasound elasticity imaging (UEI) has been found to be an available tool for characterizing mechanical changes of the implanted scaffold with high resolution and substantial detecting depth, but it is at expenses of higher cost, more specific hardware, and not easily accessible to most research groups [17, 18]. In our study, we attempted to establish a noninvasive, comprehensive, and convenient bone repair monitoring system based on diagnostic ultrasound. Appropriate scaffolds capable of providing suitable structural and biological constructs are of great importance for cellular ingrowth [19]. In our preliminary study, an injectable thermo sensitive hydrogel composite based on CS, HA, and Col was demonstrated with great biocompatibility and excellent osteogenesis performance [20, 21]. In current research, we reshaped it as a mineralized collagen fiber reinforced solid scaffold (nHAC/CS) to obtain a stable initial mechanical strength. Moreover, to explore how rBMSCs affect the bone repair process, we added rBMSCs into the scaffold and made the comparison with the simple nHAC/CS group. The ultimate goal is to innovatively excavate diagnostic ultrasound to monitor the real-time remodeling information for the long cultivation period of the two scaffold groups. Systematic in vivo indexes were extracted from ultrasonic images, such as bone mass, BMD, calcification rate, degradation rate, and uniformity of inner structure, and were compared with the analyzed indexes of DXA. The feasibility of diagnostic ultrasound was illustrated as a direct tool to evaluate the evolution of constructs online for tissue engineers.
2. Material and Method 2.1. Fabrication of Scaffold Materials 2.1.1. Preparation of Thiolated Chitosan. Chitosan (800 mg) was dissolved in acetum solution (400 mL, 1%). Iminothiolane hydrochloride (80 mg) was added after stirring for 5 hours. The pH of the solution was adjusted to 6.0 by adding
BioMed Research International sodium hydroxide (5 M). Dialysis with hydrogen chloride (5 M) was repeated 3 times. Thiolated-chitosan sample was prepared after freeze-drying. 2.1.2. Synthesis of nHAC/CS Scaffold. The synthesis of nHA/Col (nHAC) powder has been reported previously [22]. It was assembled with nanofibrils of mineralized collagen and sterilized by X-ray irradiation. Thiolated-chitosan sample (200 mg) was dissolved in sodium hydroxide (10 mL, 0.1 M) and nHAC powder (200 mg) was added into the solution. Then the solution was stirred and dispersed evenly by ultrasonic wave. We removed the solution into 96-well plates carefully, and the sample was freeze-dried at room temperature. 2.2. Cell Isolation and Culture. Bone marrow was obtained from 12-week-old male SD rats. Briefly, femurs were aseptically removed and broken. The Bone marrow was absorbed by an injector, and then rat mesenchymal stem cells (rBMSCs) were isolated to the culture flask after centrifugation at 1500 RPM for 5 min. The rBMSCs were cultured in DMEM/F12 medium and allowed to adhere for 24 hours. Nonadherent cells were then removed. After that, the cells were cultured at 37∘ C in 95% humidity and 5% carbon dioxide, and the medium was changed regularly every 3 days. After 3 weeks, adherent cells were detached by trypsin-EDTA (0.5 to 0.2 g/L, Invitrogen) and used for the in vivo experiments. 2.3. Implantation Experiment in SD Rats. All the animals were operated in the light of the guidelines for animal experiments. In this study, 18 healthy SD female rats (150 g on average), supplied by the Animal Research Center of Guangdong Province, were divided into two groups equally (A, B). After induction with midazolam, the rats were anesthetized by the 0.3 mL/kg mixture of xylazine and ketamine (2 : 1). Then the rats were placed in the prone position, depilated, and sterilized from arcus costarum to hip joint. An incision was made close to erector spinae. We performed blunt dissection on superficial fascia and created three muscular pockets in the back. For each rat, two scaffolds of the same type were implanted. The columnar scaffold (nHAC/CS) was implanted with 0.5 mL concentrated solution of rBMSCs (5 × 106 ) in the rats of group A, and in group B the same scaffold was implanted together with 0.5 mL normal saline (NS) as a control. The administration of antibiotics as prophylactic measure was carried out. All animals survived to the designated time without any major complications. The design of the study was displayed in Figure 1. 2.4. Ultrasonic Examination. Ultrasound images were taken with an ALOKA prosound 𝛼-10 premier diagnostic ultrasound system (1.1 mechanical index, 80 transmission gain) equipped with a 12 MHZ probe for all scans. Each group was performed a detection at week 0, week 1, week 2, week 4, week 6, week 8, week 10, and week 12. Rats were anesthetized in prone position with the inspection area exposed. Then we put the probe above the muscular pockets in order to observe the evolution of the scaffold constructs.
BioMed Research International Isolation of BMSCs
3 Cultivation of BMSCs
Centrifuge
SD rats
DMEM/F-12
NS
BMSCs
Skin preparation
Two muscular pockets scaffold implanation (n = 18)
Group A (n = 9)
Group B (n = 9)
Rats were sacrificed in three batches (n = 6) at weeks 4, 8, and 12
Ultrasound images analysis
DXA examination
Ultrasonic examination 0\1st\2nd\4th\6th\8th\10th\12th week
Figure 1: Schematic showing design of the study.
2.5. Ultrasound Images Analysis. The ultrasonic backscattered signal is displayed as a gray-scale array with values ranging from 0 to 255, and 0 denotes a negligible difference in resistance from the surrounding medium; the development of an ultrasound signal over time was interpreted as an increase in stiffness that may due to the solidifying development of materials. Gray-scale value, calcification rate, degradation rate, and homogeneous degree were measured and BMD was estimated by analyzing ultrasound images. 2.6. DXA. The rats were sacrificed in three batches (𝑛 = 6) at weeks 4, 8, and 12 with their affiliated tissue constructs harvested. And then each scaffold was scanned twice by a Lunar Prodigy DXA bone densitometer (GE Healthcare, Madison, WI, USA). BMD was used to evaluate the scaffolds’ ability of heterotopic osteogenesis, which can be analyzed by LunarenCORE software (ver. 10.0, standard-array mode). All the measurements were executed by the same technologist who had received professional training. 2.6.1. Gray-Scale Value. The gray-scale value (GV) was analyzed by measuring the mean GV of the implant area over time by the method of histogram echo intensity. The measurements from the six images were averaged together for each implant, reported as mean ± standard deviation. 2.6.2. Calcification Rate and Degradation Rate. All images were analyzed by ImageJ software to measure calcification rate and degradation rate. Implant region was set as region of interest (ROI). According to the GV (“Min”-“Max”) of material tested in one hour after operation, GV ranging from
“Max” to 255 was regarded as the region of calcification and the other was noncalcified region. Similarly gray-scale value ranging from 0 to “Min” and “Max” to 255 was regarded as the region of degradation and the other was no degradation region. Thus the calcification rate and degradation rate were estimated. 2.6.3. Homogeneous Degree. Implant region was set as ROI. Homogeneous degree was calculated by applying the index “kurtosis” in ImageJ. 2.6.4. BMD Estimation. According to the BMD and GV of radius, femur, tibia, pelvis, 7th cervical vertebrae, and 1st, 2nd, and 3rd lumbar vertebra in rats, regression curve was calculated. On the basis of this curve, BMD corresponding with each GV was estimated by the software of Origin 8.0. Finally, we carried out agreement analysis between estimated BMD and actual measured BMD by DXA. 2.7. Statistics. The correlation between two continuous variables, GV by ultrasound and BMD by DXA, was quantified with a Pearson correlation coefficient. Bland-Altman plots were used to assess the agreements between estimated BMD by ultrasound and actual measured BMD by DXA. The regression of the average and the difference between the two indicators were analyzed. All experimental data were reported as the mean ± standard deviation (𝑛 = 6). Levene homoscedasticity test and independent-samples 𝑡test were used to identify any significant differences between the different groups. A 𝑃 value of
Research Article Application of Ultrasound on Monitoring the Evolution of the Collagen Fiber Reinforced nHAC/CS Composites In Vivo Yan Chen,1 Yuting Yan,2 Xiaoming Li,3 He Li,2 Huiting Tan,2 Huajun Li,2 Yanwen Zhu,2 Philipp Niemeyer,4 Matin Yaega,4 and Bo Yu5 1
Department of Ultrasonic Diagnosis, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China The Second Clinical Medical College of Southern Medical University, Guangzhou 510282, China 3 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, China 4 Department of Orthopaedic surgery and Traumatology, Freiburg University Hospital, Freiburg, Germany 5 Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China 2
Correspondence should be addressed to Xiaoming Li; [email protected] and Bo Yu; [email protected] Received 31 October 2013; Accepted 4 March 2014; Published 14 April 2014 Academic Editor: Xiaowei Li Copyright © 2014 Yan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To date, fiber reinforce scaffolds have been largely applied to repair hard and soft tissues. Meanwhile, monitoring the scaffolds for long periods in vivo is recognized as a crucial issue before its wide use. As a consequence, there is a growing need for noninvasive and convenient methods to analyze the implantation remolding process in situ and in real time. In this paper, diagnostic medical ultrasound was used to monitor the in vivo bone formation and degradation process of the novel mineralized collagen fiber reinforced composite which is synthesized by chitosan (CS), nanohydroxyapatite (nHA), and collagen fiber (Col). To observe the impact of cells on bone remodeling process, the scaffolds were planted into the back of the SD rats with and without rat bone mesenchymal stem cells (rBMSCs). Systematic data of scaffolds in vivo was extracted from ultrasound images. Significant consistency between the data from the ultrasound and DXA could be observed (𝑃 < 0.05). This indicated that ultrasound may serve as a feasible alternative for noninvasive monitoring the evolution of scaffolds in situ during cell growth.
1. Introduction Cell-based bone tissue engineering has emerged as a promising alternative to traditional bone graft treatment [1]. Due to mineralized collagen fibers making up the microstructure of natural bone tissue [2], a biomimetic nanohydroxyapatite/collagen (nHA/Col) scaffold reinforced by mineralizing type I collagen fiber seems to be a very promising system for bone tissue engineering [3–5]. Hence the development of mineralized collagen fiber composites is in urgent need of a noninvasive, quantifiable, and systematic method to monitor the complex regenerate function and degradation process in vivo and in real time. Accurate in vivo data is needed for a complete understanding of the mineralized collagen fiber and guiding the scaffold design. Developing a simple and easy-to-use method to monitor regeneration process of the scaffolds is critical for bone tissue
engineering research. Current approaches for acquiring precise bone mineral density (BMD) value are mostly by dualenergy X-ray absorptiometry (DXA) or computed tomography (CT) [6]. However, for one thing, the scanning and image reconstruction procedures are of complex operations, in high consumption and with strong radiation. Thus it is difficult to meet the need of a long-term evaluation of dynamic tracking. For another, DXA and CT could only provide morphological information and are unable to achieve the exploration of bone microstructure, which is the determining factor of bone function independently of BMD [7, 8]. To date, measurements on bone constructs and some desired mechanical parameters mainly rely on destructive and timeconsuming histological and biochemical assay [9]. The substantial animal use, low repeatability, and difficulty of in vivo examination limit its application. There are also some budding nondestructive technologies. MR elastography (MRE)
2 could make an assessment of mechanical properties, but it is limited by a poor spatial resolution at 5 mm [10]. Optical coherence tomography (OCT), with high spatial resolution but low penetration capacity, was mainly used in evaluation of vascular scaffold currently [11]. Microcomputed tomography (𝜇CT) could evaluate the scaffolds systematically, but its use was limited by its high expense and equipment requirement [12]. Hence, there remains a significant need for noninvasive techniques to sequentially monitor the progress of tissue construct evolution in vivo without periodic animal sacrifice. Since the report about ultrasonic speed and attenuation in bone in 1975 [13], ultrasound was gradually developed to be a noninvasive, nonradiative diagnostic tool of bone. Physical parameters tested by ultrasound were capable of reflecting bone density, quality, and some other mechanical factors of cancellous bone [14, 15]. Consequently, ultrasonic technology has been widely used in analyzing children’s bone condition and osteoporosis in recent years and reveals a promising application [16]. However, ultrasound is rarely reported to evaluate scaffolds in bone tissue engineering. Recently, ultrasound elasticity imaging (UEI) has been found to be an available tool for characterizing mechanical changes of the implanted scaffold with high resolution and substantial detecting depth, but it is at expenses of higher cost, more specific hardware, and not easily accessible to most research groups [17, 18]. In our study, we attempted to establish a noninvasive, comprehensive, and convenient bone repair monitoring system based on diagnostic ultrasound. Appropriate scaffolds capable of providing suitable structural and biological constructs are of great importance for cellular ingrowth [19]. In our preliminary study, an injectable thermo sensitive hydrogel composite based on CS, HA, and Col was demonstrated with great biocompatibility and excellent osteogenesis performance [20, 21]. In current research, we reshaped it as a mineralized collagen fiber reinforced solid scaffold (nHAC/CS) to obtain a stable initial mechanical strength. Moreover, to explore how rBMSCs affect the bone repair process, we added rBMSCs into the scaffold and made the comparison with the simple nHAC/CS group. The ultimate goal is to innovatively excavate diagnostic ultrasound to monitor the real-time remodeling information for the long cultivation period of the two scaffold groups. Systematic in vivo indexes were extracted from ultrasonic images, such as bone mass, BMD, calcification rate, degradation rate, and uniformity of inner structure, and were compared with the analyzed indexes of DXA. The feasibility of diagnostic ultrasound was illustrated as a direct tool to evaluate the evolution of constructs online for tissue engineers.
2. Material and Method 2.1. Fabrication of Scaffold Materials 2.1.1. Preparation of Thiolated Chitosan. Chitosan (800 mg) was dissolved in acetum solution (400 mL, 1%). Iminothiolane hydrochloride (80 mg) was added after stirring for 5 hours. The pH of the solution was adjusted to 6.0 by adding
BioMed Research International sodium hydroxide (5 M). Dialysis with hydrogen chloride (5 M) was repeated 3 times. Thiolated-chitosan sample was prepared after freeze-drying. 2.1.2. Synthesis of nHAC/CS Scaffold. The synthesis of nHA/Col (nHAC) powder has been reported previously [22]. It was assembled with nanofibrils of mineralized collagen and sterilized by X-ray irradiation. Thiolated-chitosan sample (200 mg) was dissolved in sodium hydroxide (10 mL, 0.1 M) and nHAC powder (200 mg) was added into the solution. Then the solution was stirred and dispersed evenly by ultrasonic wave. We removed the solution into 96-well plates carefully, and the sample was freeze-dried at room temperature. 2.2. Cell Isolation and Culture. Bone marrow was obtained from 12-week-old male SD rats. Briefly, femurs were aseptically removed and broken. The Bone marrow was absorbed by an injector, and then rat mesenchymal stem cells (rBMSCs) were isolated to the culture flask after centrifugation at 1500 RPM for 5 min. The rBMSCs were cultured in DMEM/F12 medium and allowed to adhere for 24 hours. Nonadherent cells were then removed. After that, the cells were cultured at 37∘ C in 95% humidity and 5% carbon dioxide, and the medium was changed regularly every 3 days. After 3 weeks, adherent cells were detached by trypsin-EDTA (0.5 to 0.2 g/L, Invitrogen) and used for the in vivo experiments. 2.3. Implantation Experiment in SD Rats. All the animals were operated in the light of the guidelines for animal experiments. In this study, 18 healthy SD female rats (150 g on average), supplied by the Animal Research Center of Guangdong Province, were divided into two groups equally (A, B). After induction with midazolam, the rats were anesthetized by the 0.3 mL/kg mixture of xylazine and ketamine (2 : 1). Then the rats were placed in the prone position, depilated, and sterilized from arcus costarum to hip joint. An incision was made close to erector spinae. We performed blunt dissection on superficial fascia and created three muscular pockets in the back. For each rat, two scaffolds of the same type were implanted. The columnar scaffold (nHAC/CS) was implanted with 0.5 mL concentrated solution of rBMSCs (5 × 106 ) in the rats of group A, and in group B the same scaffold was implanted together with 0.5 mL normal saline (NS) as a control. The administration of antibiotics as prophylactic measure was carried out. All animals survived to the designated time without any major complications. The design of the study was displayed in Figure 1. 2.4. Ultrasonic Examination. Ultrasound images were taken with an ALOKA prosound 𝛼-10 premier diagnostic ultrasound system (1.1 mechanical index, 80 transmission gain) equipped with a 12 MHZ probe for all scans. Each group was performed a detection at week 0, week 1, week 2, week 4, week 6, week 8, week 10, and week 12. Rats were anesthetized in prone position with the inspection area exposed. Then we put the probe above the muscular pockets in order to observe the evolution of the scaffold constructs.
BioMed Research International Isolation of BMSCs
3 Cultivation of BMSCs
Centrifuge
SD rats
DMEM/F-12
NS
BMSCs
Skin preparation
Two muscular pockets scaffold implanation (n = 18)
Group A (n = 9)
Group B (n = 9)
Rats were sacrificed in three batches (n = 6) at weeks 4, 8, and 12
Ultrasound images analysis
DXA examination
Ultrasonic examination 0\1st\2nd\4th\6th\8th\10th\12th week
Figure 1: Schematic showing design of the study.
2.5. Ultrasound Images Analysis. The ultrasonic backscattered signal is displayed as a gray-scale array with values ranging from 0 to 255, and 0 denotes a negligible difference in resistance from the surrounding medium; the development of an ultrasound signal over time was interpreted as an increase in stiffness that may due to the solidifying development of materials. Gray-scale value, calcification rate, degradation rate, and homogeneous degree were measured and BMD was estimated by analyzing ultrasound images. 2.6. DXA. The rats were sacrificed in three batches (𝑛 = 6) at weeks 4, 8, and 12 with their affiliated tissue constructs harvested. And then each scaffold was scanned twice by a Lunar Prodigy DXA bone densitometer (GE Healthcare, Madison, WI, USA). BMD was used to evaluate the scaffolds’ ability of heterotopic osteogenesis, which can be analyzed by LunarenCORE software (ver. 10.0, standard-array mode). All the measurements were executed by the same technologist who had received professional training. 2.6.1. Gray-Scale Value. The gray-scale value (GV) was analyzed by measuring the mean GV of the implant area over time by the method of histogram echo intensity. The measurements from the six images were averaged together for each implant, reported as mean ± standard deviation. 2.6.2. Calcification Rate and Degradation Rate. All images were analyzed by ImageJ software to measure calcification rate and degradation rate. Implant region was set as region of interest (ROI). According to the GV (“Min”-“Max”) of material tested in one hour after operation, GV ranging from
“Max” to 255 was regarded as the region of calcification and the other was noncalcified region. Similarly gray-scale value ranging from 0 to “Min” and “Max” to 255 was regarded as the region of degradation and the other was no degradation region. Thus the calcification rate and degradation rate were estimated. 2.6.3. Homogeneous Degree. Implant region was set as ROI. Homogeneous degree was calculated by applying the index “kurtosis” in ImageJ. 2.6.4. BMD Estimation. According to the BMD and GV of radius, femur, tibia, pelvis, 7th cervical vertebrae, and 1st, 2nd, and 3rd lumbar vertebra in rats, regression curve was calculated. On the basis of this curve, BMD corresponding with each GV was estimated by the software of Origin 8.0. Finally, we carried out agreement analysis between estimated BMD and actual measured BMD by DXA. 2.7. Statistics. The correlation between two continuous variables, GV by ultrasound and BMD by DXA, was quantified with a Pearson correlation coefficient. Bland-Altman plots were used to assess the agreements between estimated BMD by ultrasound and actual measured BMD by DXA. The regression of the average and the difference between the two indicators were analyzed. All experimental data were reported as the mean ± standard deviation (𝑛 = 6). Levene homoscedasticity test and independent-samples 𝑡test were used to identify any significant differences between the different groups. A 𝑃 value of
